Immunological cross-reactivity and neutralization of the principal toxins of Naja sumatrana and related cobra venoms by a Thai polyvalent antivenom (Neuro Polyvalent Snake Antivenom)
Graphical abstract
Introduction
In Asia, cobra (Naja sp.) bites constitute the major cause of high mortality and morbidity associated with snake envenomation (Warrell, 2010, World Health Organization, 2010). Progressive descending paralysis and local necrosis are the prominent neurological manifestation of cobra envenomation. The early signs of muscular paralysis usually begin with ptosis, diplopia, ophthalmoplegia, dysphasia and dysphagia, followed by limb weakness and areflexia, eventually progress to respiratory failure as a consequence of intercostal muscles and diaphragm paralysis (Ahmed et al., 2008, Alirol et al., 2010). Respiratory failure occurs in 10–50% of cases (Punde, 2005, Wongtongkam et al., 2005) and is the leading cause of death in cobra envenomation (Wongtongkam et al., 2005).
Antivenom therapy has hitherto been the only definitive treatment for snake envenomation. Unfortunately, numerous studies have reported that most of the existing commercially available Asiatic cobra antivenoms are generally low in potency (Balde et al., 2013, Kularatne et al., 2009). The low neutralizing potency of Asiatic cobra antivenom (typically <2 mg venom/mL antivenom) implies that a very large amount (>20 vials) of the antivenom may be required to treat severe cobra envenomation, as the amount of venom injected by a cobra in a bite may range from 100 to 600 mg (Mirtschin et al., 2006). Administration of a very large amount of antivenom is, however, undesirable as it not only results in a very high treatment cost but also greatly increases the risk of hypersensitivity.
The present study was undertaken as part of an effort to investigate factors limiting the neutralizing and cross-neutralizing potency of Asiatic cobra antivenom. Cross-neutralization of Naja venoms by heterologous antivenom is a well-known phenomemon (Cham et al., 2013, Kornhauser et al., 2013, Leong et al., 2012a). Neuro Polyvalent Snake Antivenom (NPAV) (produced against the venoms of the Thai monocled cobra, Naja kaouthia, and three other Thai elapid snakes) was chosen for this study as it is a commonly used polyvalent antivenom in Thailand and Malaysia, and extensive cross-neutralization data of this antivenom against venoms from various cobras are available for comparison purpose (Leong et al., 2012a). This study investigated the immunoreactivity and neutralization profiles of NPAV against various principal toxins of Naja sumatrana (Equatorial spitting cobra), Naja kaouthia (Monocled cobra) and Naja sputatrix (Javen spitting cobra) venoms. Findings from the current study may provide clues to formulation of a more potent and efficacious cobra antivenom. This is particularly relevant for the optimization of paraspecific antivenom use on species like N. sumatrana (a Category 1 medically important snake in the region), the bite from which unfortunately has no species-specific antivenom available as treatment. Besides, the findings will also reveal if there is a good correlation between immunoreactivity and neutralization potency of cobra antivenom, as earlier studies have demonstrated that immunoreactivity of an antivenom does not necessarily reflect the in vivo neutralizing potency of the antivenom (Casewell et al., 2010).
Section snippets
Animals
Albino mice (ICR strain, 20–25 g) were supplied by the Laboratory Animal Centre, Faculty of Medicine, University of Malaya. The animals were handled according to the guidelines given by CIOMS on animal experimentation (Howard-Jones, 1985). The experimental protocols for all animal studies were approved by the Animal Care and Use Committee (ACUC) of the Faculty of Medicine, University of Malaya (Ethics Reference no. 2013-06-07/MOL/R/FSY). The institutional ethics committee was informed about the
Isolation of the principal toxins of N. sumatrana venom and neurotoxins from N. kaouthia and N. sputatrix venoms
The chromatographic profiles of N. sumatrana venom and its principal toxins (not shown) are similar to those reported by Yap et al. (2014). Six principal toxins were isolated from N. sumatrana venom, including one short-chain neurotoxin (sumatrana SNTX), one long-chain neurotoxins (sumatrana LNTX-II), two phospholipases A2 (sumatrana PLA2-I and sumatrana PLA2-II), and two cardiotoxins (sumatrana CTX-I and sumatrana CTX-II) (Yap et al., 2014).
Fig. 1 shows the isolation of N. kaouthia long-chain
Isolation of the cobra venom toxins
Yap et al. (2014) reported that eight of the N. sumatrana venom toxins constituted the major toxins of the venom, accounting for >90% of the total venom proteins. We selected the six most abundant toxins for this study. These include the two types of cobra venom α-neurotoxins: long and short-chain neurotoxins, and two each of the venom’s phospholipases A2 and cardiotoxins. The short-chain neurotoxin belongs to the type I α-neurotoxin subfamily, comprising 60–62 amino acid residues, while the
Conclusions
Our results suggest that there are major challenges to improving the potency of cobra antivenom, as the limitations to neutralization potency include both immunological characteristics of cobra venom toxins as well as non-immunological issues. The main cobra venom 3-finger toxins (α-neurotoxins and cardiotoxins) are generally poorly neutralized by the antivenom and poorly immunogenic too. The possible means to improve the effectiveness of neutralization include improving the avidity of
Acknowledgements
This work was supported by UM High Impact Research Grant UM.C/625/1/HIR/MOE/E00040-20001 from the Ministry of Education Malaysia, UMRG RG282-14AFR from University of Malaya, Malaysia and Science Fund, MOSTI, Government of Malaysia.
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2019, Journal of ProteomicsCitation Excerpt :As with the acidic PLA2 from N. kaouthia and N. naja venoms, the major PLA2 of N. philippinensis venom was found to be non-lethal up to 5 μg/g in mice, a dose that was ~25-time higher than the venom LD50 (0.18 μg/g) in this study. In contrast, the basic and neutral venom PLA2 of spitting cobras N. sputatrix [16] and N. sumatrana [59], respectively, are known to be lethal (LD50 = 0.5–2.0 μg/g). The biological activity of the acidic PLA2 could be associated with local effect (venom ophthalmia and tissue necrosis), and potentiation of cytotoxic activity for defensive as well as digestive purposes [5,60].